Project Summary Because of the link between quorum sensing (QS) and virulence in challenging pathogens like the Gram-negative bacterium Pseudomonas aeruginosa, there is significant interest in characterizing and inhibiting key proteins that enable this method of bacterial communication. LasR, a regulator protein with a central role in mediating QS response in P. aeruginosa, is a promising target for inhibition. Because most work with LasR has been carried out at the cellular level, a number of assumptions have been made about the biochemical events that are likely to involve this transcriptional regulator. The overall objectives of this project are to provide a more complete mechanistic understanding of LasR and related QS regulator proteins and use this characterization to inform the design of novel inhibitors with therapeutic relevance. The first aim will measure kinetic and thermodynamic parameters describing LasR dimerization and DNA recognition to determine the likely relevant intermediates leading to the formation of an active LasR-DNA complex. The initial steps in LasR recognition of its autoinducer will also be modeled with in vitro systems. The second aim will examine how the previously identified oxidative stress response by LasR correlates with its recognition of multiple promoters. Covalent modification of multiple cysteines involved in this redox response will be explored as a new route to LasR inhibition. The third aim involves the preparation and evaluation of inhibitors designed to target the LasR dimerization interface. These approaches to inhibitor design offer advantages that previously reported inhibitors do not since neither strategy requires competition of the inhibitor with the native autoinducer. Both in vitro assays with purified LasR and cell-based reporter assays will be used in evaluating inhibitors. This proposed project is likely to provide detailed information about LasR and its inhibition at the biomolecular level that can be leveraged toward the development of therapeutically impactful antimicrobial strategies against P. aeruginosa and related pathogens. Additionally, undergraduate researchers will be central to data collection, interpretation and reporting on this project, providing them with the opportunity to gain significant biomedical research exposure.